Method and composition for preventing and treating solid tumors

ABSTRACT

Compositions and methods of preventing or treating a breast cancer are disclosed. The composition and method can utilize an endothelin B agonist and a chemotherapeutic agent as active ingredients to treat a solid tumor in mammals, including humans. Alternatively, the composition and method can utilize an endothelin B antagonist and an optional angiogenesis inhibitor to treat a solid tumor in mammals, including humans.

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims the benefit of U.S. provisional patentapplication No. 60/420,960, filed Oct. 24, 2002.

FIELD OF THE INVENTION

[0002] The present invention relates to the prevention and treatment ofsolid tumors, such as breast tumors, in a mammal, either byadministration of therapeutically effective amounts of an endothelinagonist and a chemotherapeutic drug, or by administration of atherapeutically effective amount of an endothelin antagonist.

BACKGROUND OF THE INVENTION

[0003] Although the present specification is directed primarily tobreast tumors, the invention disclosed and claimed herein can be used inthe treatment and prevention of solid tumors in general, as set forthhereafter.

[0004] Breast cancer incidence has increased substantially in the last10 years, and is the single leading cause of death for women ages 40-49years in the United States. In 2001, 192,000 cases and 40,000 deathsmade breast cancer the most common cancer, after superficial skincancers, and the second leading cause of cancer death (Lacey et al.,Environ Mol Mutagen, 39(2-3):82-88 (2002)).

[0005] The development of a breast cancer is a complex process involvinga combination of factors, such as environmental and genetic factors. Oneextensively studied breast tumor model is the chemically induced ratmammary carcinogenesis model (Refs. 9, 18, 19, 39, 54). Chemicallyinduced mammary tumorigenesis in rats is the model most closelyresembling a human cancer (40).

[0006] Chemically induced rat mammary carcinogenesis typically isachieved by administration of 7,12-dimethylbenzene(a)anthracene (DMBA)(37) or N-methylnitrosourea (MNU) (37). Tumors induced by DMBA or MNUhave different morphological characteristics. In particular, tumorsinduced by MNU are more localized at the breast and are less likely tometastasize (25). Therefore, MNU often is chosen as the chemical agentfor the specific induction of breast tumors in rats. These breast tumorscan be benign with fibroadenomas and papillomas, or they can bemalignant (54). Rats have six pairs of mammary glands, one in thecervical region, two in the thoracic region, one in the abdominalregion, and two in the ingual region (4, 54). Virgin rats treated withMNU develop more tumors in the thoracic region than the abdominal region(41).

[0007] The development of tumor vasculature has been studiedextensively. Tumors greater than a few millimeters in size require aconstant nutrient supply, and, therefore, have their own vascular bedand blood flow (10). Recruitment of new vasculature from preexistingblood vessels is termed “angiogenesis.” Without constant nourishmentfrom these developing blood vessels, the tumors become hypoxic andsubsequently die. Therefore, tumor vasculature has been a target ofcancer therapy for a considerable time (10).

[0008] Tumor blood vessels develop substantially differently from normalvasculature, and have different properties. Single layered epithelialcells are the first hastily formed tumor blood vessels. It has beensuggested that these blood vessels are recruited when the tumor secretescertain growth factors, like vascular endothelial growth factor (VEGF),in response to hypoxic conditions (23). These newly formed tumor bloodvessels do not have a smooth muscle layer or innervation (29, 36, 57).

[0009] Tumors also incorporate mature blood vessels that possess alltheir autoregulatory functions (29). Normal tissue vascular tone isgoverned by a host of endogenous factors like H⁺, K⁺, Ca²⁺, pO₂, pCO₂,nitric oxide (NO), as well as other regulatory substances likeendothelin (ET-1) (24, 46).

[0010] ET-1 is a potent vasoconstrictor and contributes significantly inregulating vascular tone (61). In breast cancer tissue, ET_(B) receptorsare found on stromal fibroblast cells (5, 34). Endothelins have beenfound to be mitogenic to fibroblasts (53), melanocytes, vascular smoothmuscle, and endothelium (3, 35, 52). Investigators have shown anincrease in ET-1, ET-3, and ET_(B) receptor expression in breastcarcinomas (1). It has been shown that both ET-1 and ET-3 cause anincrease in VEGF, which is an important angiogenic factor (35). Thus, anincrease in ET-1 promotes tumor growth. Several studies have reported anincrease in ET-1 levels in breast tumors (1, 21, 31, 33, 59, 60).

[0011] The present invention is directed to the effect of endothelinantagonists and endothelin agonists on systemic hemodynamics and bloodcirculation in solid tumor tissues. The present invention also isdirected to the use of endothelin agonists and endothelin antagonists inthe treatment of solid tumors.

SUMMARY OF THE INVENTION

[0012] The present invention is directed to administration oftherapeutically effective amounts of an endothelin agonist and achemotherapeutic agent to an individual in need thereof in the treatmentof a solid tumor. The present invention also is directed toadministration of a therapeutically effective amount of an endothelinantagonist to an individual in need thereof in the prevention andtreatment of a solid tumor, such as a breast tumor.

[0013] In particular, tumors need a blood supply to grow. ET is apowerful regulator of blood flow. ET_(A) receptors have been found to bevasoconstrictors, and ET_(B) receptors have been found to bevasodilators. In accordance with the present invention, it has beendemonstrated that breast tumor tissue has abundant ET_(B) receptors, andthat an ET_(B) receptor antagonist can block the increased blood flow tobreast tumor tissue induced by ET-1. Accordingly, an endothelinantagonist, particularly an ET_(B) receptor antagonist, is useful toprevent the growth of breast or other solid tumors having ET_(B)receptors regulating their blood flow.

[0014] In addition, because ET_(B) receptors are vasodilators, it hasbeen found that an ET_(B) receptor agonist, in combination with achemotherapeutic agent, is useful in the treatment of a solid tumor,such as those found in breast cancer. In this embodiment, the ET_(B)receptor agonist more effectively delivers the chemotherapeutic agent tothe breast tumor resulting in an enhanced treatment.

[0015] Accordingly, one aspect of the present invention is to provide amethod of treating solid tumors comprising administering to a mammal inneed thereof a therapeutically effective amount of an endothelin agonistand a chemotherapeutic agent.

[0016] Another aspect of the present invention is to provide acomposition comprising an endothelin agonist, in particular an ET_(B)agonist. The composition is useful in the treatment of solid tumors. Theendothelin agonist is used in conjunction with a chemotherapeutic agent.In particular, the present invention also is directed to compositionscontaining an endothelin agonist, and to methods of administering theendothelin agonist, in conjunction with a chemotherapeutic agent, totreat solid tumors.

[0017] Still another aspect of the present invention is to provide acomposition comprising an endothelin agonist, a second therapeutic agentuseful in the treatment of a solid tumor, and an excipient.

[0018] Still another aspect of the present invention is to provide amethod of preventing or treating solid tumors comprising administeringto a mammal in need thereof a therapeutically effective amount of anendothelin antagonist. The endothelin antagonist can be an endothelin Bantagonist or a mixed endothelin A/B antagonist. Preferably, theendothelin antagonist comprises a specific endothelin B (ET_(B))antagonist. The endothelin antagonist optionally is used in conjunctionwith an angiogenesis inhibitor, radiation treatment, or both.

[0019] Another aspect of the present invention is to provide acomposition comprising an endothelin antagonist, in particular an ET_(B)antagonist, to an individual in need thereof. The composition is usefulin the prevention and treatment of solid tumors.

[0020] Another aspect of the present invention is to provide acomposition comprising an endothelin antagonist, a second therapeuticagent useful in the prevention or treatment of a solid tumor, and anexcipient.

[0021] Yet another aspect of the present invention is to provide anarticle of manufacture for human pharmaceutical use, comprising (a) acontainer, and (b1) a packaged composition comprising an endothelinagonist and, optionally, (b2) a packaged composition comprising a secondtherapeutic agent useful in the treatment of a solid tumor, and (c) apackage insert containing directions for use of the composition orcompositions administered simultaneously or sequentially, in thetreatment of a solid tumor. In a preferred embodiment, the endothelinagonist is an ET_(B) receptor agonist and the second therapeutic agentis a chemotherapeutic agent.

[0022] Another aspect of the present invention is to provide an articleof manufacture for human pharmaceutical use, comprising (a) a container,(b1) a packaged composition comprising an endothelin antagonist and,optionally, (b2) a packaged composition comprising a second therapeuticagent useful in the treatment of a solid tumor, and (c) a package insertcontaining directions for use of the composition or compositions,administered simultaneously or sequentially, in the prevention ortreatment of a solid tumor. In a preferred embodiment, the endothelinantagonist is an ET_(B) receptor antagonist, and the second therapeuticagent is an angiogenesis inhibitor, radiation treatment, or both.

[0023] These and other novel aspects of the present invention willbecome apparent from the following detailed description of the preferredembodiments of the invention taken in conjunction with the figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024]FIG. 1 contains bar graphs showing the effect of ET-1 on systemichemodynamics of saline-treated and MNU-treated, tumor-bearing rats;

[0025]FIG. 2 contains bar graphs showing the effect of ET-1 on bloodflow and regional vascular resistance in the breast tissue ofsaline-treated and MNU-treated rats;

[0026]FIG. 3 contains plots showing the effect of ET-1 on perfusion,CMBC, and velocity of blood cells in breast tissue of saline-treated andtumor tissue of MNU-treated rats;

[0027]FIG. 4 contains plots showing the effect of BQ788 on ET-1-inducedchanges in blood perfusion, CMBC, and velocity of blood cells in breasttissue of saline-treated and tumor tissue of MNU-treated rats; and

[0028]FIG. 5 contains plots showing the effect of IRL1620 onpaclitaxel-induced changes in tumor perfusion.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The present invention is directed to compositions and methods ofpreventing and treating solid tumors, including breast tumors. Inparticular, the present invention is directed to pharmaceuticalcompositions comprising either (a) an endothelin agonist and,optionally, a chemotherapeutic agent or (b) an endothelin antagonist,and optionally, angiogenesis inhibitor.

[0030] The present invention also is directed to articles of manufacturecomprising an endothelin antagonist and an optional angiogenesisinhibitor, packaged separately or together, and an insert havinginstructions for using these active agents to prevent or treat a solidtumor.

[0031] In addition, the present invention is directed articles ofmanufacture comprising an endothelin agonist and a chemotherapeuticagent, packaged separately or together, and an insert havinginstructions for using these active agents to treat a solid canceroustumor.

[0032] One method disclosed herein utilizes an endothelin agonist and achemotherapeutic agent in the treatment of a solid tumor. The agonistand chemotherapeutic agent can be administered in sufficient amounts,simultaneously or sequentially, to achieve the desired therapeuticeffect.

[0033] Another method disclosed herein utilizes an endothelinantagonist, optionally with an angiogenesis inhibitor, in the treatmentof solid tumors. The antagonist and angiogenesis inhibitor can beadministered in sufficient amounts, simultaneously or sequentially, toachieve the desired effect.

[0034] For the purposes of the invention disclosed herein, the term“treatment” includes preventing, retarding the progression of,shrinking, or eliminating a solid tumor. As such, the term “treatment”includes both medical therapeutic and/or prophylactic administration, asappropriate.

[0035] The term “container” means any receptacle and closure thereforsuitable for storing, shipping, dispensing, and/or handling apharmaceutical product.

[0036] The term “insert” means information accompanying a pharmaceuticalproduct that provides a description of how to administer the product,along with the safety and efficacy data required to allow the physician,pharmacist, and patient to make an informed decision regarding use ofthe product. The package insert generally is regarded as the “label” fora pharmaceutical product.

[0037] The term “prodrug” means compounds that transform rapidly in vivoto a compound useful in the invention, for example, by hydrolysis. Athorough discussion of prodrugs is provided in Higuchi et al., Prodrugsas Novel Delivery Systems, Vol. 14, of the A.C.S.D. Symposium Series,and in Roche (ed.), Bioreversible Carriers in Drug Design, AmericanPharmaceutical Association and Pergamon Press, 1987.

[0038] Endothelin is a vasoactive substance known to modulate blood flowand also has mitogenic properties. Endothelin is present in largeconcentrations in breast carcinoma tissues compared to normal breasttissue. In accordance with the present invention, it has been shown thata subtype of endothelin receptor (ET_(B)) also is increased in breastcancer. Endothelin acts on ET_(B) receptors to produce vascular dilationand increase in blood flow to the breast tumor tissue. Importantly, italso has been found that an ET_(B) receptor antagonist can block theincrease in tumor blood flow induced by endothelin.

[0039] Because endothelin and ET_(B) receptors are overexpressed inbreast cancer, a selective ET_(B) receptor antagonist, e.g., BQ788, canbe used to block endothelin-induced vasodilation in the breast tumortissue, and cut off or reduce the blood supply and nutrient supplyneeded for the breast tumor to grow. An ET_(B) antagonist can be usedalone, or in combination with an angiogenesis inhibitor, likethalidomide, that inhibits the formation of new blood vessels in thetumor tissue. Once the blood supply and nutrient supply to the tumortissue are reduced, the growth of the tumor also is reduced.

[0040] In addition, most chemotherapeutic agents have cytotoxicproperties that are targeted to destroy cancer cells, but in the processinflict considerable damage to the body's normal physiological systems.It would be of great advantage, therefore, to selectively deliverchemotherapeutic agents to the tumor tissue. Accordingly, an ET_(B)receptor agonist that selectively increases blood supply to the tumorcan increase the delivery and efficacy of the chemotherapeutic agent.Therefore, ET_(B) receptor agonists can selectively increase thedelivery of chemotherapeutic agents, like tamoxifen, to a breast tumorand increase efficacy of the chemotherapeutic agent.

[0041] More particularly, tumor blood supply has become a target ofcancer therapy. Several vasoactive substances are known to modulateblood flow including endothelin-1 (ET-1). ET-1 is present in largeconcentrations in breast carcinoma tissues (i.e., 11.95 pg/mg tissue)compared to normal breast tissue (i.e., 0.12 pg/mg tissue) (Kojima etal., Surg. Oncol., 4(6):309-315 (1995); Kurbel et al., Med. Hypotheses,52(4):329-333 (1999); Patel et al., Mol. Cell Endocrinol.,126(2):143-151 (1997); Yamashita et al., Cancer Res., 52(14):4046-4049(1992); Yamashita et al., Res. Commun. Chem. Pathol. Pharmacol.,74(3):363-369 (1991).

[0042] Studies have shown that ET-1, ET-3, and ET_(B) receptorexpression is increased in breast cancer (grade III, strong stainingcompared to negative staining in controls) (Alanen et al.,Histopathology, 36(2):161-167 (2000)). It also has been found that ET-1produces an increase in blood flow to the breast tumor by stimulatingET_(B) receptors. BQ788, an ET_(B) receptor antagonist, completelyblocked ET-1 induced increase in tumor blood flow. Because breast tumortissue has enhanced ET_(B) receptor expression, an ET_(B) receptorantagonist can be used to selectively decrease breast tumor bloodsupply, and an ET_(B) receptor agonist can be used to increase bloodflow to the breast tumor tissue.

[0043] Accordingly, an ET_(B) receptor agonist in combination with achemotherapeutic agent decreases breast tumor growth. In addition, anET_(B) receptor antagonist, either alone or in combination with anangiogenesis inhibitor, significantly decreases the breast tumor growth.

[0044] Administration of an ET_(B) receptor agonist in combination witha chemotherapeutic agent also can be used to treat or prevent othersolid tumors, including, but not limited to, ovarian cancer, coloncarcinoma, Kapoli's sarcoma, breast cancer, and melanomas. An endothelinantagonist, alone or in combination with an angiogenesis inhibitor, alsocan be used in the treatment and prevention of solid tumors.

[0045] The following table lists the ET receptor expression for varioussolid tumors. Tumor ET receptor expression References Ovarian cancerET_(A) and ET_(B) receptors Bagnato et al., Cancer Res, 1999, 59,720-727 Colon carcinoma ET_(A) receptors are present Egidy et al., Am Jin stroma Pathology, 2000, 157, ET_(B) receptors in 1863-1874endothelium and myofibroblasts Kaposi's sarcoma ET_(A) and ET_(B)receptors in Bagnato et al., Am J tumor and intratumoral Pathol, 2001,158, vessels 841-847 Breast cancer ET_(B) receptors Alanen et al.,Histopathology: 2000: 36(2): 161 Melanoma ET_(B) receptors Kikuchi etal., Biochem Biophys Res Comm, 1996, 219, 734-739

[0046] In one embodiment of the present invention, a solid tumor istreated using an endothelin agonist in conjunction with achemotherapeutic agent. In this method, the endothelin agonist, notablyan ET_(B) agonist, increases blood flow in the breast tumor, which isrich in ET_(B) receptors. The ET_(B) agonist, therefore, provides a moreselective target for the chemotherapeutic agent and improves thechemotherapeutic effect of the agent.

[0047] ET_(B) agonists useful in the present invention include, but arenot limited, to, ET-1, ET-2, ET-3, BQ3020, IRL1620, sarafotoxin S6c,[Ala ^(1, 3, 11, 15)]ET-1, and mixtures thereof.

[0048] It is theorized, but not relied upon herein, that endothelinagonists stimulate ET_(B) receptors and dilate tumor blood vessels,thereby increasing delivery of the chemotherapeutic agent to the tumor.Endothelin agonists also increase blood perfusion of the solid tumor,and thereby increase oxygenation of the tissue. Improved oxygenation isknown to enhance the therapeutic action of chemotherapeutic agents. Themitogenic action of endothelin also can help increase the action ofchemotherapeutic agents, when administered together. The mitogenicaction of an endothelin agonist can improve incorporation ofchemotherapeutic agents in the dividing cells, and increase the efficacyof the chemotherapeutic agents.

[0049] In this embodiment, the ET_(B) agonist is used in conjunctionwith a chemotherapeutic agent. The ET_(B) agonist enhances thetherapeutic benefit of chemotherapy treatment, including inductionchemotherapy and primary (neoadjuvant) chemotherapy. In addition,chemotherapy is frequently indicated as an adjuvant to surgery in thetreatment of a cancer. The goal of chemotherapy in the adjuvant settingis to reduce the risk of recurrence and enhance disease-free survivalwhen the primary tumor has been controlled. Chemotherapy is utilized asa treatment adjuvant for a cancer, frequently when the disease ismetastatic. An ET_(B) agonist, therefore, is particularly usefulfollowing surgery in the treatment of a solid tumor in combination withchemotherapy.

[0050] Chemotherapeutic agents that can be used in the present methodinclude, but are not limited to, alkylating agents, antimetabolites,hormones and antagonists thereof, radioisotopes, antibodies, as well asnatural products, and mixtures thereof. For example, an ET_(B) agonistcan be administered with antibiotics, such as doxorubicin and otheranthracycline analogs, nitrogen mustards, such as cyclophosphamide,pyrimidine analogs such as 5-fluorouracil, cisplatin, hydroxyurea, taxoland its natural and synthetic derivatives, and the like. As anotherexample, in the case of mixed tumors, such as adenocarcinoma of thebreast, where the tumors include gonadotropin-dependent andgonadotropin-independent cells, the ET_(B) agonist can be administeredin conjunction with leuprolide or goserelin (synthetic peptide analogsof LH-RH). Examples of chemotherapeutic agents useful in the method ofthe present invention are listed in the following table. Alkylatingagents Nitrogen mustards mechlorethamine cyclophosphamide ifosfamidemelphalan chlorambucil Nitrosoureas carmustine (BCNU) lomustine (CCNU)semustine (methyl-CCNU) Ethylenimine/Methylmelamine thriethylenemelamine(TEM) triethylene thiophosphoramide (thiotepa) hexamethylmelamine (HMM,altretamine) Alkyl sulfonates busulfan Triazines dacarbazine (DTIC)Antimetabolites Folic Acid analogs methotrexate trimetrexate Pyrimidineanalogs 5-fluorouracil fluorodeoxyuridine gemcitabine cytosinearabinoside (AraC, cytarabine) 5-azacytidine 2,2′-difluorodeoxycytidinePurine analogs 6-mercaptopurine 6-thioguanine azathioprine2′-deoxycoformycin (pentostatin) erythrohydroxynonyladenine (EHNA)fludarabine phosphate 2-chlorodeoxyadenosine (cladribine, 2-CdA) Type ITopoisomerase Inhibitors camptothecin topotecan irinotecan Naturalproducts Antimitotic drugs paclitaxel Vinca alkaloids vinblastine (VLB)vincristine vinorelbine Taxotere ® (docetaxel) estramustine estramustinephosphate Epipodophylotoxins etoposide teniposide Antibioticsactimomycin D daunomycin (rubidomycin) doxorubicin (adriamycin)mitoxantroneidarubicin bleomycinsplicamycin (mithramycin) mitomycinCdactinomycin Enzymes L-asparaginase Biological response modifiersinterferon-alpha IL-2 G-CSF GM-CSF Differentiation Agents retinoic acidderivatives Radiosensitizers metronidazole misonidazoledesmethylmisonidazole pimonidazole etanidazole nimorazole RSU 1069 EO9RB 6145 SR4233 nicotinamide 5-bromodeozyuridine 5-iododeoxyuridinebromodeoxycytidine Miscellaneous agents Platinium coordination complexescisplatin carboplatin Anthracenedione mitoxantrone Substituted ureahydroxyurea Methylhydrazine derivatives N-methylhydrazine (MIH)procarbazine Adrenocortical suppressant mitotane (o,p′-DDD)ainoglutethimide Cytokines interferon (α, β, γ) interleukin-2 Hormonesand antagonists Adrenocorticosteroids/ antagonists prednisone andequivalents dexamethasone ainoglutethimide Progestinshydroxyprogesterone caproate medroxyprogesterone acetate megestrolacetate Estrogens diethylstilbestrol ethynyl estradiol/ equivalentsAntiestrogen tamoxifen Androgens testosterone propionatefluoxymesterone/equivalents Antiandrogens flutamidegonadotropin-releasing hormone analogs leuprolide Nonsteroidalantiandrogens flutamide Photosensitizers hematoporphyrin derivativesPhotofrin ® benzoporphyrin derivatives Npe6 tin etioporphyrin (SnET2)pheoboride-a bacteriochlorophyll-a naphthalocyanines phthalocyanineszinc phthalocyanines

[0051] Examples of chemotherapeutic agents that are particularly usefulin conjunction with an ET_(B) agonist include, for example, adriamycin,camptothecin, carboplatin, cisplatin, daunorubicin, doxorubicin,interferon (alpha, beta, gamma), interleukin 2, irinotecan, docetaxel,paclitaxel, topotecan, and therapeutically effective analogs andderivatives of the same.

[0052] In another embodiment of the present invention, an endothelinantagonist utilized in the method and composition can be any ET_(B)receptor antagonist known in the art. ET_(B) receptors are potentvasodilators. ET_(B) antagonists inhibit the activity of ET_(B), and areused to restrict blood flow.

[0053] ET_(B) antagonists useful in the present invention can beselective ET_(B) antagonists or balanced ET_(A)/ET_(B) antagonists.ET_(B) receptor antagonists, and balanced ET_(A)/ET_(B) antagonists,useful in the treatment and/or prevention of solid tumors are set forthin Appendices A through C herein. Additional useful endothelinantagonists can be found in U.S. Patent Application Publication No. US2002/0082285 A1, incorporated herein by reference.

[0054] Examples of ET_(B) antagonists useful in the present inventioninclude, but are not limited to, atrasentan, tezosentan, bosentan,sitaxsentan, enrasentan, Ro468443, TBC10950, TBC10894, A192621, A308165,SB209670, SB217242, A182086, (s)-Lu302872, J-104132, TAK-044,Sarafotoxin 56c, IRL2500, RES7011, Aselacins A, B, and C, Ro470203,Ro462005, sulfamethoxazole, cochinmicin I, II, and III, L749329,L571281, L754142, J104132, CGS27830, A182086, PD142893, PD143296,PD145065, PD156252, PD159020, PD160672, PD160874, TM-ET-1, IRL3630,Ro485695, L753037, LU224332, PD142893, LU302872, PD145065, Ro610612,SB217242, BQ788, and mixtures thereof. BQ-788 is a preferred specificendothelin B antagonist, and is the sodium salt ofN-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methylleucyl-D-1-methoxycarbonyltriptophanyl-DNIe (see Proc. Natl. Acad. Sci. USA, 91:4892-4896 (1994)).

[0055] In addition to a conventional endothelin antagonist, a compoundthat inhibits the formation of endogenous endothelin also can be used asthe endothelin antagonist in the present invention. Such compounds areuseful because they prevent endothelin formation and, therefore,decrease the activity of endothelin receptors. One class of suchcompounds is the endothelin converting enzyme (ECE) inhibitors. UsefulECE inhibitors include, but are not limited to, CGS34225 (i.e.,N-((1-((2-(S)(acetylthio)-1-oxopentyl)-amino)-1-cyclopentyl)-carbonyl-S-4-phenylphenyl-alaninemethyl ester) and phosphoramidon (i.e.,N-(1-rhamnopyranosyloxyhydroxyphosphinyl)-Leu-Trp).

[0056] As discussed more fully hereafter, the ET_(B) receptor antagonistcan be used in conjunction with an angiogenesis inhibitor. As previouslystated, angiogenesis is the generation of new vasculature frompreexisting blood vessels. An angiogenesis inhibitor retards oreliminates the generation of new vasculature.

[0057] Any angiogenesis inhibitor known in the art can be used with anET_(B) antagonist in the present method. Examples of angiogenesisinhibitors include, but are not limited to, thalidomide, marimastat,COL-3, BMS-275291, squalamine, 2-ME, SU6668, neovastat, Medi-522,EMD121974, CAI, celecoxib, interleukin-12, IM862, TNP470, avastin,gleevac, herceptin, and mixtures thereof.

[0058] In one method of the present invention, wherein an ET_(B)antagonist and an optional angiogenesis inhibitor are administered to anindividual in need thereof to treat a solid tumor by restricting bloodflow and inhibiting the formation of new vasculature, the individualalso can be treated using radiation therapy and/or a radiosensitizer.

[0059] The term “radiosensitizer,” as used herein, is defined as acompound administered to a human or other animal in a therapeuticallyeffective amount to increase the sensitivity of cells to electromagneticradiation and/or to promote the treatment of diseases that are treatablewith electromagnetic radiation. Radiosensitizers can be administered inconjunction with an ET_(B) antagonist and optional angiogenesisinhibitor.

[0060] The terms “electromagnetic radiation” and “radiation” as usedherein include, but are not limited to, radiation having the wavelengthof 10-20 to 100 meters. Preferred embodiments of the present inventionemploy the electromagnetic radiation of gamma-radiation (10-20 to 10-13m), X-ray radiation (10-12 to 10-9 m), ultraviolet light (10 nm to 400nm), visible light (400 nm to 700 nm), infrared radiation (700 nm to 1.0mm), and microwave radiation (1 mm to 30 cm).

[0061] Many cancer treatment protocols currently employ radiosensitizersactivated by electromagnetic radiation, e.g., X-rays. Examples ofX-ray-activated radiosensitizers include, but are not limited to, thefollowing: metronidazole, misonidazole, desmethylmisonidazole,pimonidazole, etanidazole, nimorazole, mitomycin C, RSU 1069, SR 4233,EO9, RB 6145, nicotinamide, 5-bromodeoxyuridine (BUdR),5-iododeoxyuridine (IUdR), bromodeoxycytidine, fluorodeoxyuridine(FUdR), hydroxyurea, cisplatin, and therapeutically effective analogsand derivatives of the same.

[0062] Photodynamic therapy (PDT) of cancers employs visible light asthe radiation activator of the sensitizing agent. Examples ofphotodynamic radiosensitizers include, but are not limited to,hematoporphyrin derivatives, PHOTOFRIN®, benzoporphyrin derivatives,NPe6, tin etioporphyrin (SnET2), pheoborbide-a, bacteriochlorophyll-a,naphthalocyanines, phthalocyanines, zinc phthalocyanine, andtherapeutically effective analogs and derivatives of the same.

[0063] In summary, the structure, growth, and function of the bloodvessels in breast tumors are markedly different from that of normalbreast tissue due to changes in the production of growth factors, likevascular endothelial growth factor (VEGF), vasoactive substances likeendothelin-1 (ET-1), and cytokines. The role of ET-1 in breast tumorangiogenesis is not adequately understood. Studies have shown that theexpression of proET-1, proET-3, and ET_(B) receptors is increased inbreast tumor. However, it is unclear whether there is any change in ET-1induced vascular responses in the breast tumor. Hence, the systemichemodynamics and regional circulatory effects of ET-1 in rats withbreast tumors was investigated.

[0064] For the first time, it has been demonstrated that ET-1 producesan increase in blood flow to the breast tumor by stimulating ET_(B)receptors. BQ788, an ET_(B) receptor antagonist, completely blocked anET-1 induced increase in tumor blood flow. Because breast tumor tissuehas enhanced ET_(B) receptor expression, an ET_(B) receptor antagonistcan be used to decrease blood supply selectively to tumor tissue.

[0065] Similarly, an ET_(B) receptor agonist increases blood supply totumor tissue, thereby facilitating administration of a chemotherapeuticdrug to the tumor. Accordingly, an ET_(B) receptor agonist can be usedin combination with a chemotherapeutic agent in the treatment of a solidtumor, like a breast tissue. In addition, most chemotherapeutic agentshave cytotoxic properties and are targeted to destroying cancer cells.However, in the process, chemotherapeutic agents inflict considerabledamage to the body's normal physiological systems. ET_(B) receptoragonists that selectively increase blood supply to the tumor thereforecan increase the delivery and efficacy of chemotherapeutic agents.

[0066] ET_(B) receptor antagonists can be used in the treatment of abreast cancer either alone or in combination with an angiogenesisinhibitor. Angiogenesis inhibitors prevent the formation of new bloodvessels needed for the growth of the tumor. Therefore, a combination ofan angiogenesis inhibitor with an ET_(B) receptor antagonist, whichselectively decreases the blood supply to breast tumor tissue,significantly decreases tumor growth.

[0067] Therefore, an ET_(B) receptor agonist in combination with achemotherapeutic agent decreases solid tumor growth. In addition, anET_(B) receptor antagonist, either alone or in combination with anangiogenesis inhibitor, significantly decreases solid tumor growth.

[0068] Materials and Methods

[0069] Animals

[0070] Female Sprague Dawley rats (Harlan Co., Madison, Wis.) weighing180-200 grams (g) were used. All animals were housed, three to a cage,in a temperature controlled room (23±1° C.), humidity (50±10%), andartificial light (0600-1800 hr). The animals were given food and waterad libitum. The experiments were conducted after the animals had beenacclimatized to the environment for at least four days.

[0071] Drugs

[0072] N-methylnitrosourea (MNU) was purchased from Ash Stevens Inc.,Detroit, Mich. BQ788(N-cis2,6-dimethylpiperidinocarbonyl-L-gamma-methylleucyl-D-1-methoxycarbonyltrptophanyl-D-Nle),IRL1620, and Endothelin-1 (ET-1) were obtained from American PeptideCompany Inc., Sunnyvale, Calif. BQ788 was dissolved in saline and ET-1was dissolved in 0.1% albumin.

[0073] Methods for Effect of IRL1620 and Taxol on Breast Tumor Perfusion

[0074] MNU (50 mg/kg, i.p.) or saline (1 ml/kg, i.p.) was administeredto female Sprague Dawley rats. After the tumors reached 2-4 cm indiameter, the blood flow experiments were performed. The animals weredivided into the following groups:

[0075] (i) Saline injection followed by taxol (3 mg/kg) after 15 minutesin normal rats (N=4);

[0076] (ii) IRL 1620 (3 nmol/kg) injection followed by taxol (3 mg/kg)after 15 minutes in normal rats (N=4);

[0077] (iii) Saline injection followed by taxol (3 mg/kg) after 15minutes in tumor bearing rats (N=4); and

[0078] (iv) IRL 1620 (3 nmol/kg) injection followed by taxol (3 mg/kg)after 15 minutes in tumor bearing rats (N=4).

[0079] Surgical Preparations

[0080] Rats were anesthetized with urethane (1.5 g/kg, i.p.) (SigmaChemicals, St. Louis, Mo.). The left femoral vein was cannulated (PE 50tubing, Clay Adams, Parsipanny, N.J.) for drug administration. The leftfemoral artery was cannulated, and was used for withdrawal of referenceblood samples. The right femoral artery was cannulated and connected toa Gould P23 ID pressure transducer for recording the blood pressure on aGrass P7D polygraph (Grass Instrument Co., Quincy, Mass., USA) through a7PI preamplifier. The heart rate (HR) was recorded through a 7P4B Grasstachograph (Grass Instrument Co., Quincy, Mass.) triggered from bloodpressure signals.

[0081] Breast Blood Perfusion Measurement by Laser Doppler Flowmetry(LDF)

[0082] The blood perfusion to the mammary gland of the rats was measuredusing laser Doppler flowmetry. The animals were shaved around thenipples and the skin surrounding the mammary glands was dissected out. Astandard model fiber optic probe was secured to the mammary artery andconnected to a Periflux PF2b 4000 Laser Doppler Flowmetry (Perimed KB,Stockholm, Sweden). The time constant was set to 1.5 seconds, and theband width was set to 4 KHz.

[0083] Statistical Analysis

[0084] All data are presented as mean±SEM. Data were analyzed usinganalysis of variance followed by Duncan's test. A level of p<0.05 wasconsidered significant.

[0085] RESULTS

[0086] Effect of IRL1620 and Taxol on Breast Tumor Perfusion

[0087] No change in blood flow to the breast tissue of normal rats wasobserved following the administration of saline or IRL1620 and taxol.Significant differences were observed between the blood flow in thetumor tissue after IRL1620 injection (36.3%, p<0.05) and after taxoladministration (51.9%, p<0.0-5) from baseline (see FIG. 5).

[0088] Effect of IRL 1620 and Taxol on Blood Pressure

[0089] No change in blood pressure was observed following theadministration of saline or IRL 1620 and taxol in normal and tumorbearing rats.

[0090] Experimental Protocol for ET-1 Infusion Into Rats

[0091] The following groups of animals were studied to evaluate theeffect of ET-1 infusion on systemic hemodynamics and blood flow to themammary tissue of normal and tumor-bearing rats.

[0092] (i) ET-1 (50 ng/kg/min) infusion for 30 minutes in rats treatedwith saline (N=6); and

[0093] (ii) ET-1 (50 ng/kg/min) infusion for 30 minutes in treated withMNU (50 mg/kg, i.p.) (N=6).

[0094] The following groups were studied to evaluate the role of ET_(B)receptors on the changes induced by ET-1 infusion on the systemichemodynamics and blood flow to the mammary tissue of normal rats andrats with breast tumors:

[0095] (i) BQ788 (0.5 pmol/kg) infusion for 20 minutes followed by ET-1(50 ng/kg/min) infusion for 30 minutes in rats treated with saline(N=5);

[0096] (ii) BQ788 (0.5 pmol/kg) infusion for 20 minutes followed by ET-1(50 ng/kg/min) infusion for 30 minutes in rats treated with MNU (50mg/kg, i.p.) (N=5).

[0097] MNU and saline treatments were performed as intraperitoneal(i.p.) injections three months prior to the study. Rats were palpatedregularly starting four weeks after the treatments. Once tumors reachedan optimal size (i.e., 4-8 mm in diameter), the experiments wereinitiated. Systemic hemodynamic and regional circulation parameters weredetermined at baseline, 30, 60, and 120 minutes after starting ET-1 (50ng/kg/min) infusion. Because ET-1 infusion was performed for 30 minutes,the 30-minute data shows the effect of ET-1, and the 60- and 120-minutedata indicates duration of the ET-1 effect.

[0098] Surgical Preparations

[0099] Rats were anesthetized with urethane (1.5 g/kg, i.p.) (SigmaChemicals, St. Louis, Mo.). All surgical areas were shaved and cleanedwith alcohol swabs. The left femoral vein was cannulated (PE 50 tubing,Clay Adams, Parsipanny, N.J.) for drug administration. The left femoralartery was cannulated (PE 50 tubing) and was used for withdrawal ofreference blood sample in microsphere studies using a withdrawal pump(Model 22, Harvard Apparatus, South Natick, Mass.). The right femoralartery was cannulated (PE 50 tubing) and connected to a Gould P23 IDpressure transducer for recording the blood pressure on a Grass P7Dpolygraph (Grass Instrument Co., Quincy, Mass., USA) through a 7PIpreamplifier. The heart rate (HR) was recorded through a 7P4B Grasstachograph (Grass Instrument Co., Quincy, Mass.) triggered from bloodpressure signals. The right carotid artery was exposed and a PE 50tubing was guided through the common carotid artery into the leftventricle. The presence of the cannula in the left ventricle wasconfirmed by recording the pressure on the Grass polygraph using theStatham P23 DC pressure transducer (Grass Instrument Co., Quincy,Mass.). When the cannula reached the left ventricle; the diastolicpressure dropped to zero. In order to maintain the blood pO₂, pCO₂, andpH constant, and to avoid the effect of respiration on blood pressureand HR, animals were kept on constant rate artificial respiration byinserting an endotracheal cannula connected to a rodent ventilator(Model 683, Harvard Apparatus Inc., South Natick, Mass.).

[0100] Determination of Systemic Hemodynamics and Regional Circulation

[0101] Systemic hemodynamics and regional blood circulation weredetermined using a literature described procedure (13, 16, 47). At eachmeasurement, a thoroughly mixed suspension of approximately 100,000microspheres (15±1 μm diameter) labeled with ⁴⁶Sc (scandium), ¹¹³Sn(tin), ¹⁴¹Ce (cerium), or ⁹⁵Nb (niobium) (New England NuclearCorporation, Boston, Mass., USA) in 0.2 ml saline were injected into theleft ventricle and flushed with 0.3 ml saline over a 15 second period.In order to calculate blood flow, arterial blood was withdrawn at a rateof 0.5 ml/min through the right femoral artery. Blood was withdrawn for90 seconds starting about 5-10 seconds before microsphere injection. Atthe end of the experiment, the animals were sacrificed with an overdoseof pentobarbital sodium. All tissues and organs were dissected out,weighed, and placed in vials. The radioactivity in the standards, theblood samples, and the tissue samples were counted in a Packard MinaxiAuto-Gamma 5000 series gamma counter (Packard Instruments Co., DownersGrove, Ill.) with preset windows discriminating the isotope energies.The following parameters were calculated: (1) cardiac output (CO)((radioactivity injected x withdrawal rate of arterialblood)/radioactivity in sampled arterial blood), (2) stroke volume (SV)(CO/HR), (3) total peripheral resistance (TPR) (mean arterial pressure(MAP)/CO), (4) regional blood flow ((radioactivity in tissue xwithdrawal rate of arterial blood)/radioactivity in sampled arterialblood), and (5) regional vascular resistance (MAP/regional blood flow).The data were calculated using computer programs described in theliterature (45).

[0102] Breast Blood Perfusion Measurement by Laser Doppler Flowmetry(LDF)

[0103] Blood perfusion to the mammary gland of the rats was measuredusing laser Doppler flowmetry as described in literature procedures (50,51). The animals were shaved around the nipples. The skin surroundingthe mammary glands was dissected out as a lambeau about 6 cm wide and 4cm long. A standard model fiber optic probe was applied to the surfaceof the lambeau, and secured to the tissue by double stick tape. Thelambeau was placed in a metal holder and taped down to prevent movement,then connected to a Periflux PF2b 4000 Laser Doppler Flowmetry (PerimedKB, Stockholm, Sweden). The time constant was set at 1.5 seconds and thebandwidth was set at 4 KHz.

[0104] Statistical Analysis

[0105] All data are presented as mean±SEM. Data were analyzed usinganalysis of variance followed by Duncan's test. A level of p<0.05 wasconsidered significant.

[0106] RESULTS

[0107] Effect of ET-1 on Systemic Hemodynamics in Normal andTumor-Bearing Rats

[0108] The baseline systemic hemodynamic parameters in normal (salinetreated) rats were MAP: 111.1±4.8 mmHg; CO:268.6±17.6 ml/min;SV:0.87±0.06 ml; TPR:419.6±24.37 mmHg.min/ml; and HR:312.5±20.2beats/min. In normal rats, a significant increase in MAP was observed at30 minutes (14.5%; p<0.05), and a decrease at 120 minutes (17.8%;p<0.05) following ET-1 infusion. TPR increased at 120 minutes (49.2%;p<0.05). CO decreased at 60 and 120 minutes (22.9% and 42.5%respectively; p<0.05) after ET-1 infusion. SV decreased at 60 and 120minutes (20.9% and 36% respectively; p<0.05). No significant change inHR was observed (FIG. 1).

[0109] The baseline systemic hemodynamic parameters in tumor-bearing(MNU treated) rats were similar to that in normal rats. A significantincrease in MAP was observed at 30 minutes (19.1%; p<0.05) and at 60minutes (15.3%; p<0.05) following ET-1 infusion in tumor-bearing rats.TPR increased at 30 minutes (73.9%; p<0.05), 60 minutes (39.7%; p<0.05),and 120 minutes (71.4%; p<0.05) following administration of ET-1. COdecreased at 30, 60 and 120 minutes (29.4%, 16.7% and 36.1%respectively; p<0.05). SV decreased significantly at 30, 60 and 120minutes (31.1%, 17.9% and 32.1% respectively; p<0.05). No change in HRwas observed (FIG. 1).

[0110] Effect of ET-10n Regional Blood Flow and Vascular Resistance inthe Breast Tissue of Normal and Tumor-Bearing Rats

[0111] No change in blood flow to the breast tissue of normalsaline-treated rats was observed following the administration of ET-1. Asignificant decrease (18.61%; p<0.05) in vascular resistance at 60minutes was observed, which is 30 minutes post ET-1 infusion, in thebreast tissue of normal rats (FIG. 2).

[0112] Significant differences were observed between the blood-flow andthe regional vascular resistance in the breast tissue of tumor-bearing(MNU treated) and normal (saline treated) rats. A significant increase(153%; p<0.05) in blood flow to the breast tissue of tumor-bearing ratsas compared to normal rats was observed at 60 minutes followingadministration of ET-1. The vascular resistance in the tumor-bearingrats was significantly different at baseline (102%; p<0.05) and at 60minutes (147%; p<0,05) following ET-1 administration compared to normalrats.

[0113] Effect of ET-1 on Blood Perfusion in the Breast Tissue of Normaland TumorBearing Rats as Measured by LDF

[0114]FIG. 3 shows the changes in perfusion, concentration of movingblood cells (CMBC), and velocity of red blood cells (RBC) in the breasttissue of tumor-bearing and normal rats. Blood perfusion in the breasttissue of normal rats did not change after ET-1 administration.Perfusion in the breast tissue of tumor-bearing rats at 30 minutesfollowing ET-1 administration increased significantly (176%; p<0.05)compared to normal rats. This increase in perfusion returned to baselineat 60 and 120 minutes following ET-1 administration in tumor-bearingrats.

[0115] The CMBC in tumor-bearing rats increased significantly (54%;p<0.05) at 60 minutes post ET-1 administration as compared to normalrats. CMBC returned to baseline at 120 minutes after ET-1administration. The velocity of RBC increased significantly (252%;p<0.05) at 30 minutes post ET-1 administration compared to normal rats.Two hours (120 minutes) after ET-1 administration, the velocity of RBCin tumor-bearing rats returned to baseline.

[0116] Effect of BQ7880n ET-1 Induced Changes in Blood Perfusion in theBreast Tissue of Normal and Tumor-Bearing Rats as Measured by LDF

[0117]FIG. 4 shows the effect of BQ788 on changes induced by ET-1 inblood perfusion, CMBC, and velocity of RBC in tumor-bearing and normalrats, respectively. Blood perfusion in the breast tissue of normal ratsdid not change significantly after BQ788 administration or ET-1infusion. However, perfusion in the breast tumor tissue of tumor-bearingrats decreased significantly at 30 (25.25±5.7%; P<0.05) and 60 minutes(25.17±2.8%; P<0.05) following ET-1 infusion in BQ788 pretreated rats.Pretreatment with BQ788 attenuated the increase in perfusion induced byET-1 in tumor-bearing rats. No difference between the perfusion inbreast tissue of tumor-bearing rats and normal rats was observedfollowing ET-1 administration in BQ788 pretreated rats.

[0118] The baseline CMBC in tumor-bearing rats was significantly higherthan the baseline CMBC of breast tissue of normal rats (42.4%; P<0.05).However, after BQ788 infusion, no difference between CMBC oftumor-bearing and normal rats was observed. In addition, no differencein velocity of RBC between the two groups was observed.

[0119] The above tests show the effect of ET-1 on systemic hemodynamicsand blood flow to the breast tissue of saline-treated and MNU-treatedtumor-bearing rats. It is known that ET-1 stimulates angiogenesis bypromoting production of VEGF. Studies have shown that ET-1 is increasedin many cancer tissues like breast carcinoma (60), breast phyllode tumor(59), prostate carcinoma (31), liver carcinoma (21), and somemeningiomas (33). The above tests demonstrate changes in ET-1-inducedvascular responses in the breast tumor. The method used in these testswas a well-established radioactive microsphere technique to study thesystemic hemodynamics and regional blood circulation (12-15).

[0120] ET-1 is a powerful vasoconstrictor (61). ET-1 belongs to a familyof peptides approximately 21 amino acids long. At least three forms ofET receptors exist, and are known as ET_(A), ET_(B), and ET_(C). ET_(A)has a higher affinity for ET-1, but ET_(B) has equal affinity for bothET-1 and ET-3 (2, 17, 42). ET-1 has complex cardiovascular effects. Whenadministered to anesthetized and ventilated rats, an immediate decreasefollowed by a sustained increase in blood pressure is observed (22). Ithas been found that ET_(A) receptors are responsible for thevasoconstrictor responses, and ET_(B) receptors are responsible for thevasodilatory actions of ET-1. ET-1 administration resulted in anincrease in blood flow to the skin tumors possibly due to thevasodilatory actions of ET_(B) (6). Similar results in blood flow to thebreast tumor of rats are expected because of an increase in ET-1 andET_(B) in breast tumors.

[0121] Infusion of 50 ng/kg/min of ET-1 caused a biphasic response inblood pressure, i.e., an immediate but short lasting decrease followedby a sustained increase. These results are in accordance with previousstudies (20, 30, 38, 56). ET-1 produced a marked pressor response inboth normal and tumor-bearing rats, which was accompanied by asignificant decrease in SV and CO. TPR significantly increased in bothnormal and tumor-bearing rats and may explain the observed pressorresponse.

[0122] Baseline blood flow to the breast tumor tissue of tumor-bearingrats was higher than blood flow in normal animals. This was observed inan earlier study and is theorized, but not relied upon, as beingattributed to the recruitment of new blood vessels in the tumor (55).Blood flow to the breast tumor following ET-1 administration wassignificantly increased as compared to that observed in the breasttissue of normal rats. Laser Doppler flowmetry showing an increase inblood perfusion to the breast tumor confirmed an increase in blood flowobserved in the breast tumor tissue following ET-1 administration. Theincrease in blood perfusion is theorized, but not relied upon, as beingattributed to an increase in either velocity of RBC velocity or CMBC, orboth. At the end of ET-1 infusion an increase in velocity of RBC wasobserved, whereas an increase in CMBC was observed 30 minutes after ET-1infusion.

[0123] Further, the observed increase in blood flow in response to ET-1is theorized, but not relied upon, as being attributed to ET_(B)mediated vasodilation. Studies have shown that ET-1 and ET_(B) receptorexpression is augmented in the breast cancer tissue (1, 60). Inaccordance with the present invention, it was found that administrationof BQ788 blocked the ET-1-induced increase in blood flow to the tumortissue. BQ788 (i.e.,N-cis-2,6-dimethylpiperidinocarbonyl-L-gamma-methylleucyl-D-1-methoxycarbonyltrptophanyl-D-Nle)is a specific ET_(B) receptor antagonist. BQ788 inhibits binding toET_(B) receptors with an IC₅₀ value of 1.2 nM.

[0124] BQ788 was used to determine the role of ET_(B) receptors in ET-1induced vasodilation in the breast tumor. This result suggests thatET-1-induced vasodilatory responses are mediated through ET_(B)receptors. Expression of ET_(B) receptors is significantly higher in theendothelial cells than in the smooth muscle cells, and is regulated byvarious growth factors and cytokines (49). Normal breast tissue has ahigher level of ET_(B) than ET_(A) receptors (1), and it is theorized,but not relied upon, that during breast cancer, ET_(B) receptors areoverexpressed and contribute to maintaining blood flow to the tumortissue.

[0125] As tumors grow, new blood vessels are recruited to supplynutrients. This recruitment can be incorporation of existing vesselsinto the tumor or creation of new blood vessels (7). Studies have shownthat new vessels have different physical properties than normalvasculature. Unlike normal vessels, these vessels do not have any smoothmuscle layers or any innervation, but consist only of single layers ofendothelial cells.

[0126] In summary, the present tests clearly demonstrate that theinfusion of ET-1 produced an increase in blood flow and a decrease invascular resistance of the breast tumor tissue, and that this increasein blood flow can be blocked by an ET_(B) receptor antagonist, e.g.,BQ788.

[0127] The increased blood flow observed in the rat breast tumor isattributed to increased ET_(B) receptors. Therefore, blocking thesereceptors can reduce blood flow to the tumor. The clinical significanceof these findings is that ET_(B) receptor antagonists play a role inreducing blood supply to the breast tumor tissue, and thereby preventand/or reduce growth of the breast tumor, and solid tumors in general.

[0128] The test results, therefore, clearly demonstrate that ET_(B)antagonists, like BQ788, can prevent or treat solid tumors. ET_(B)antagonists optionally can be combined with an angiogenesis inhibitor topotentiate the effects of the ET_(B) antagonist.

[0129] The ET_(B) antagonist, optional angiogenesis inhibitor, ET_(B)agonist, and chemotherapeutic agent (hereafter collectively “activeingredients”) can be formulated in suitable excipients for oraladministration or for parenteral administration. Such excipients arewell known in the art. The active ingredients typically are present insuch a composition in an amount of about 0.1% to about 75% by weight.

[0130] Pharmaceutical compositions containing the active ingredients aresuitable for administration to humans or other mammals. Typically, thepharmaceutical compositions are sterile, and contain no toxic,carcinogenic, or mutagenic compounds that would cause an adversereaction when administered. Administration of the pharmaceuticalcomposition can be performed before, during, or after the onset of solidtumor growth.

[0131] A method of the present invention can be accomplished usingactive ingredients as described above, or as a physiologicallyacceptable salt, derivative, prodrug, or solvate thereof. The activeingredients can be administered as the neat compound, or as apharmaceutical composition containing either or both entities.

[0132] The active ingredients can be administered by any suitable route,for example by oral, buccal, inhalation, sublingual, rectal, vaginal,intracisternal through lumbar puncture, transurethral, nasal,percutaneous, i.e., transdermal, or parenteral (including intravenous,intramuscular, subcutaneous, and intracoronary) administration.Parenteral administration can be accomplished using a needle andsyringe, or using a high pressure technique, like POWDERJECT™.

[0133] The pharmaceutical compositions include those wherein the activeingredients are administered in an effective amount to achieve theirintended purpose. More specifically, a “therapeutically effectiveamount” means an amount effective to prevent development of, toeliminate, to retard the progression of, or to reduce the size of asolid tumor. Determination of a therapeutically effective amount is wellwithin the capability of those skilled in the art, especially in lightof the detailed disclosure provided herein.

[0134] A “therapeutically effective dose” refers to that amount of theactive ingredients that results in achieving the desired effect.Toxicity and therapeutic efficacy of such active ingredients can bedetermined by standard pharmaceutical procedures in cell cultures orexperimental animals, e.g., determining the LD₅₀ (the dose lethal to 50%of the population) and the ED₅₀ (the dose therapeutically effective in50% of the population). The dose ratio between toxic and therapeuticeffects is the therapeutic index, which is expressed as the ratiobetween LD₅₀ and ED₅₀. A high therapeutic index is preferred. The dataobtained can be used in formulating a range of dosage for use in humans.The dosage of the active ingredients preferably lies within a range ofcirculating concentrations that include the ED₅₀ with little or notoxicity. The dosage can vary within this range depending upon thedosage form employed, and the route of administration utilized.

[0135] The exact formulation, route of administration, and dosage isdetermined by an individual physician in view of the patient'scondition. Dosage amount and interval can be adjusted individually toprovide levels of the active ingredients that are sufficient to maintaintherapeutic or prophylactic effects.

[0136] The amount of pharmaceutical composition administered isdependent on the subject being treated, on the subject's weight, theseverity of the affliction, the manner of administration, and thejudgment of the prescribing physician.

[0137] Specifically, for administration to a human in the curative orprophylactic treatment of a breast tumor, oral dosages of activeingredients, individually generally are about 10 to about 200 mg dailyfor an average adult patient (70 kg), typically divided into two tothree doses per day. Thus, for a typical adult patient, individualtablets or capsules contain about 0.1 to about 50 mg active ingredients,in a suitable pharmaceutically acceptable vehicle or carrier, foradministration in single or multiple doses, once or several times perday. Dosages for intravenous, buccal, or sublingual administrationtypically are about 0.1 to about 10 mg/kg per single dose as required.In practice, the physician determines the actual dosing regimen that ismost suitable for an individual patient, and the dosage varies with theage, weight, and response of the particular patient. The above dosagesare exemplary of the average case, but there can be individual instancesin which higher or lower dosages are merited, and such are within thescope of this invention.

[0138] The active ingredients can be administered alone, or in admixturewith a pharmaceutical carrier selected with regard to the intended routeof administration and standard pharmaceutical practice. Pharmaceuticalcompositions for use in accordance with the present invention thus canbe formulated in a conventional manner using one or more physiologicallyacceptable carriers comprising excipients and auxiliaries thatfacilitate processing of the active ingredients into preparations whichcan be used pharmaceutically.

[0139] These pharmaceutical compositions can be manufactured in aconventional manner, e.g., by conventional mixing, dissolving,granulating, dragee-making, emulsifying, encapsulating, entrapping, orlyophilizing processes. Proper formulation is dependent upon the routeof administration chosen. When a therapeutically effective amount of theactive ingredients are administered orally, the composition typically isin the form of a tablet, capsule, powder, solution, or elixir. Whenadministered in tablet form, the composition can additionally contain asolid carrier, such as a gelatin or an adjuvant. The tablet, capsule,and powder contain about 5% to about 95% of an active ingredients, andpreferably from about 25% to about 90% active ingredients. Whenadministered in liquid form, a liquid carrier, such as water, petroleum,or oils of animal or plant origin, can be added. The liquid form of thecomposition can further contain physiological saline solution, dextroseor other saccharide solutions, or glycols. When administered in liquidform, the composition contains about 0.5% to about 90% by weight ofactive ingredients, and preferably about 1% to about 50% of activeingredients.

[0140] When a therapeutically effective amount of the active ingredientsis administered by intravenous, cutaneous, or subcutaneous injection,the composition is in the form of a pyrogen-free, parenterallyacceptable aqueous solution. The preparation of such parenterallyacceptable solutions, having due regard to pH, isotonicity, stability,and the like, is within the skill in the art. A preferred compositionfor intravenous, cutaneous, or subcutaneous injection typicallycontains, in addition to an isotonic vehicle.

[0141] Suitable active ingredients can be readily combined withpharmaceutically acceptable carriers well-known in the art. Suchcarriers enable the active agents to be formulated as tablets, pills,dragees, capsules, liquids, gels, syrups, slurries, suspensions and thelike, for oral ingestion by a patient to be treated. Pharmaceuticalpreparations for oral use can be obtained by adding the activeingredients with a solid excipient, optionally grinding the resultingmixture, and processing the mixture of granules, after adding suitableauxiliaries, if desired, to obtain tablets or dragee cores. Suitableexcipients include, for example, fillers and cellulose preparations. Ifdesired, disintegrating agents can be added.

[0142] The active ingredients can be formulated for parenteraladministration by injection, e.g., by bolus injection or continuousinfusion. Formulations for injection can be presented in unit dosageform, e.g., in ampules or in multidose containers, with an addedpreservative. The compositions can take such forms as suspensions,solutions, or emulsions in oily or aqueous vehicles, and can containformulatory agents such as suspending, stabilizing, and/or dispersingagents.

[0143] Pharmaceutical compositions for parenteral administration includeaqueous solutions of the active agent in water-soluble form.Additionally, suspensions of the active ingredients can be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils or synthetic fatty acid esters. Aqueousinjection suspensions can contain substances which increase theviscosity of the suspension. Optionally, the suspension also can containsuitable stabilizers or agents that increase the solubility of thecompounds and allow for the preparation of highly concentratedsolutions. Alternatively, a present composition can be in powder formfor constitution with a suitable vehicle, e.g., sterile pyrogen-freewater, before use.

[0144] The active ingredients also can be formulated in rectalcompositions, such as suppositories or retention enemas, e.g.,containing conventional suppository bases. In addition to theformulations described previously, the active ingredients also can beformulated as a depot preparation. Such long-acting formulations can beadministered by implantation (for example, subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, theactive ingredients can be formulated with suitable polymeric orhydrophobic materials (for example, as an emulsion in an acceptable oil)or ion exchange resins, or as sparingly soluble derivatives, forexample, as a sparingly soluble salt.

[0145] In particular, the active ingredients can be administered orally,buccally, or sublingually in the form of tablets containing excipients,such as starch or lactose, or in capsules or ovules, either alone or inadmixture with excipients, or in the form of elixirs or suspensionscontaining flavoring or coloring agents. Such liquid preparations can beprepared with pharmaceutically acceptable additives, such as suspendingagents. The active ingredients also can be injected parenterally, forexample, intravenously, intramuscularly, subcutaneously, orintracoronarily. For parenteral administration, the active ingredientsare best used in the form of a sterile aqueous solution which cancontain other substances, for example, salts, or monosaccharides, suchas mannitol or glucose, to make the solution isotonic with blood.

[0146] For veterinary use, the active ingredients are administered as asuitably acceptable formulation in accordance with normal veterinarypractice. The veterinarian can readily determine the dosing regimen androute of administration that is most appropriate for a particularanimal.

[0147] As stated above, it has been discovered that using an ET_(B)antagonist, alone or together with an angiogenesis inhibitor, is usefulin the treatment and prevention of solid tumors.

[0148] The angiogenesis inhibitor, like the ET_(B) antagonist, isadministered in an effective amount to perform its intended function.The angiogenesis inhibitor can be administered by any suitable means,typically using a composition containing the angiogenesis inhibitor.

[0149] The angiogenesis inhibitor can be administered simultaneouslywith the ET_(B) antagonist, or prior to or after ET_(B) antagonistadministration. The ET_(B) antagonist and optional angiogenesisinhibitor also can be administered in conjunction with radiationtreatment of the solid tumor and an optional radiosensitizer.

[0150] In another embodiment, the solid tumor can be treated byadministration of therapeutically effective amounts of an ET_(B) agonistand a chemotherapeutic agent. Administration of the ET_(B) agonist andchemotherapeutic agent can be performed as described above for theET_(B) antagonist and angiogenesis inhibitor.

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[0212] Modifications and variations of the invention as hereinbefore setforth can be made without departing from the spirit and scope thereof,and, therefore, only such limitations should be imposed as are indicatedby the appended claims.

What is claimed is:
 1. A method of treating a solid tumor comprisingadministering to a mammal in need thereof a therapeutically effectiveamount of an endothelin B agonist and a therapeutically effective amountof a chemotherapeutic agent.
 2. The method of claim 1 wherein the solidtumor is selected from the group consisting of an ovarian tumor, a colontumor, Kaposi's sarcoma, a breast tumor, a melanoma, a prostate tumor, ameningioma, a liver tumor, and a breast phyllode tumor.
 3. The method ofclaim 2 wherein the solid tumor is a breast tumor.
 4. The method ofclaim 1 wherein the endothelin agonist is selected from the groupconsisting of ET-1, ET-2, ET-3, BQ3020, IRL1620, sarafotoxin 56c,[Ala^(1, 3, 11, 15)]ET-1, and mixtures thereof.
 5. The method of claim 4wherein the endothelin B agonist comprises IRL1620.
 6. The method ofclaim 1 wherein the chemotherapeutic agent is selected from the groupconsisting of adriamycin, camptothecin, carboplatin, cisplatin,daunorubicin, doxorubicin, alpha, beta, or gamma interferon, interleukin2, irinotecan, docetaxel, paclitaxel, topotecan, and mixtures thereof.7. The method of claim 1 wherein the endothelin B agonist and thechemotherapeutic agent are administered simultaneously.
 8. The method ofclaim 7 wherein the endothelin B agonist and the chemotherapeutic agentare administered from a single composition.
 9. The method of claim 7wherein the endothelin B agonist and the chemotherapeutic agent areadministered from separate compositions.
 10. The method of claim 1wherein the endothelin B agonist and the chemotherapeutic agent areadministered sequentially.
 11. The method of claim 10 wherein thechemotherapeutic agent is administered prior to the endothelin Bagonist.
 12. The method of claim 10 wherein the endothelin B agonist isadministered prior to the chemotherapeutic agent.
 13. The method ofclaim 1 wherein the mammal is a human.
 14. A composition comprising achemotherapeutic agent, an endothelin B agonist, and an optionalexcipient.
 15. An article of manufacture comprising: (a) a packagedcomposition comprising an endothelin B agonist, and; (b) an insertproviding instructions for administration of (a) to treat a solid tumorin a mammal; and (c) a container for (a) and (b).
 16. An article ofmanufacture comprising: (a) a packaged composition comprising anendothelin B agonist; (b) a packaged composition comprising achemotherapeutic agent; (c) an insert providing instructions for asimultaneous or sequential administration of (a) and (b) to treat asolid tumor in a mammal; and (d) a container for (a), (b), and (c) 17.An article of manufacture comprising: (a) a packaged compositioncomprising an endothelin B agonist and a chemotherapeutic agent; (b) aninsert providing instructions for administration of (a) to treat a solidtumor in a mammal; and (c) a container for (a) and (b).
 18. A method oftreating a solid tumor comprising administering to a mammal in needthereof a therapeutically effective amount of an endothelin Bantagonist.
 19. The method of claim 18 wherein the solid tumor isselected from the group consisting of an ovarian tumor, a colon tumor,Kaposi's sarcoma, a breast tumor, a melanoma, a prostate tumor, ameningioma; a liver tumor, and a breast phyllode tumor.
 20. The methodof claim 19 wherein the solid tumor is a breast tumor.
 21. The method ofclaim 18 wherein the endothelin B antagonist comprises a specificendothelin B antagonist.
 22. The method of claim 18 wherein theendothelin B antagonist comprises a balanced endothelin B antagonist.23. The method of claim 18 wherein the endothelin B antagonist isselected from the group consisting of compounds 1 through 74 ofAppendices A, B, and C.
 24. The method of claim 18 wherein theendothelin B antagonist is selected from the group consisting ofcompounds 1 through 22 of Appendix A.
 25. The method of claim 18 whereinthe endothelin B antagonist is selected from the group consisting ofcompounds 23 through 32 of Appendix B.
 26. The method of claim 18wherein the endothelin B antagonist is selected from the groupconsisting of compounds 33 through 74 of Appendix C.
 27. The method ofclaim 18 wherein the endothelin B antagonist is selected from the groupconsisting of atrasentan, tezosentan, bosentan, sitaxsentan, enrasentan,Ro468443, TBC10950, TBC10894, A192621, A308165, SB209670, SB17242,A182086, (s)-Lu302872, J-104132, TAK-044, Sarafotoxin 56c, IRL2500,RES7011, Aselacins A, B, and C, Ro470203, Ro462005, sulfamethoxazole,cochinmicin I, II, and III, L749329, L571281, L754142, J104132,CGS27830, PD142893, PD143296, PD145065, PD156252, PD159020, PD160672,PD160874, TM-ET-1, IRL3630, Ro485695, L75037, LU224332, PD142893,LU302872, PD145065, Ro610612, SB217242, BQ788, and mixtures thereof. 28.The method of claim 18 wherein the endothelin B antagonist comprisesBQ788.
 29. The method of claim 18 further comprising administering atherapeutically effective amount of an angiogenesis inhibitor.
 30. Themethod of claim 29 wherein the angiogenesis inhibitor is selected fromthe group consisting of thalidomide, marimastat, COL-3, BMS275291,squalamine, 2-ME, SU6668, neovastat, Medi522, EMD121974, CAI, celecoxib,interleukin-12, IM862, TNP470, avastin, gleevac, herceptin, and mixturesthereof.
 31. The method of claim 29 wherein the endothelin B antagonistand the angiogenesis inhibitor are administered simultaneously.
 32. Themethod of claim 31 wherein the endothelin B antagonist and theangiogenesis inhibitor are administered from a single composition. 33.The method of claim 29 wherein the endothelin B antagonist and theangiogenesis inhibitor are administered from separate compositions. 34.The method of claim 29 wherein the and endothelin B antagonist and theangiogenesis inhibitor are administered sequentially.
 35. The method ofclaim 34 wherein the angiogenesis inhibitor is administered prior to theendothelin B antagonist.
 36. The method of claim 34 wherein theendothelin B antagonist is administered prior to the angiogenesisinhibitor.
 37. The method of claim 18 further comprising treating thesolid tumor with radiation and an optional radiosensitizer.
 38. Themethod of claim 37 wherein the radiosensitizer is selected from thegroup consisting of metronidazole, misonidazole, desmethylmisonidazole,pimonidazole, etanidazole, nimorazole, mitomycin C, RSU 1069, SR 4233,EO9, RB 6145, nicotinamide, 5-bromodeoxyuridine, 5-iododeoxyuridine,bromodeoxycytidine, fluorodeoxyuridine, hydroxyurea, cisplatin,therapeutically effective analogs and derivatives thereof, and mixturesthereof.
 39. The method of claim 18 wherein the mammal is a human.
 40. Acomposition comprising (a) an endothelin B antagonist, (b) anangiogenesis inhibitor, and (c) an optional excipient.
 41. An article ofmanufacture comprising: (a) a packaged composition comprising anendothelin B antagonist; (b) a packaged composition comprising anangiogenesis inhibitor; (c) an insert providing instructions for asimultaneous or sequential administration of (a) and (b) to treat asolid tumor in a mammal; and (d) a container for (a), (b), and (c). 42.An article of manufacture comprising: (a) a packaged compositioncomprising an endothelin antagonist and an angiogenesis inhibitor; (b)an insert providing instructions for administration of (a) to treat asolid tumor in a mammal; and (c) a container for (a) and (b).
 43. Anarticle of manufacture comprising: (a) a packaged composition comprisingan endothelin B antagonist; (b) an insert providing instructions foradministration of (a) to treat a solid tumor in a mammal; and (c) acontainer for (a) and (b).